1. Field of the Invention
This invention relates to a method for recovery of intracellular material by disruption of microbial cells with carbon dioxide under pressure.
2. Background Art
Microbial cells have long been recognized as an important source of commercially useful biochemicals, antibiotics, food and enzymes. With increasing demand of microbial products in industry and medicine, considerable efforts have recently been committed to develop technologies for production of intracellular microbial materials from microorganisms and various genetically altered cultures. Release of cell contents is also vital to many investigations of bacterial metabolism. In any case, the cell disruption is a necessary operation for recovery or isolation of protein, intracellular enzymes and organelles. A variety of disruption techniques had been developed, and some are available commercially. Mechanical methods appear to be favored at the present time by their economic advantages, although several non-mechanical methods, particularly the enzymatic lysis, have attracted great attention. However, many of these methods often degrade or denature proteins in the process.
Yeast is the most widely accepted traditional food item which has been exploited as a potential protein source; however, despite its nutritional value and abundant supply, the potential of yeast as a major food source has been hampered by various problems. Ideally, yeast cell biomass should be consumed directly as food or as food ingredients, but the thick cell wall reduces the digestibility and bio-availability of protein. The cell wall substances often cause allergic responses, diarrhea, and flatulence. Even after the cell wall is removed, some lipid components contribute to off-flavors by oxidation, and more importantly, the functional properties of proteins are impaired in the process. Finally, the protein from single cells contains a high level of nucleic acids, which can cause uricacidemia and gout.
The nature of the cell walls and their removal have been discussed in the literature. Autolysis (particularly, thio-activated autolysis) and enzymatic lysis are among the conventional techniques in wide use for disintegration of microbial cells, other than yeast, to recover intracellular enzymes. Although most microbial cell walls can readily be ruptured, yeast cell walls are very difficult to disintegrate. Selection of yeast strains with weaker cell walls and alkaline treatment can facilitate yeast protein recovery. Other methods, such as homogenization, freeze-thawing, and pressurization have been used to rupture the yeast cells.
Among these methods, pressurization may be the least expensive if the pressure can be kept at a reasonably low range. Prior investigation of gas pressure for rupturing cells, but not yeast cells, has found that carbon dioxide was effective at 500 psi.
Supercritical fluid (SCF) has recently demonstrated great potential in its applications to food and pharmaceutical industries. The fluid possesses a combination of "gas-like" and "liquid-like" properties. It penetrates like a gas, and functions like a liquid. Such unique characteristics of SCF have found broad applications in diverse areas, particularly for extraction and separation of natural products. Various prior studies have extensively applied SCF as a medium, in place of conventional solvents, in enzyme reactions.